The immune response to infection with Mycobacterium tuberculosis (Mtb) involves complex interactions between macrophages, T-cells, cytokines and accessory molecules. Mycobacteria evade the host’s immune response by interfering with cell mediated immune systems. Granulomas are central to the host’s defenses against Mtb. These responses may be modified by immune alterations especially in patients co-infected with Human Immunodeficiency Virus (HIV). This study investigated the immunohistochemical profile of CD4+, CD8+, CD68+, Th-17 (also known as Interleukin-17 cells) and Forkhead box (FOXP3) cells, accessory molecule expression (HLA Class I and II) and selected cytokines (Interleukin 2, 4, 6 and Interferon-) of various cell types within mycobacterial granulomas, in lymph nodes from ten HIV negative and ten HIV positive patients. Tissue from a foreign body granuloma in skin was utilised for comparison. This study illustrated retention of CD4+ lymphocyte numbers within granulomas from HIV negative (-) patients but documented a reversal in the ratio of CD4+ to CD8+cells in granulomas from HIV positive (+) patients. Similar IL-17 cell counts were noted in mycobacterial granulomas from both HIV (-) and HIV (+) patients. CD68 was identified in all macrophages and HLA Class II stained 100% of cells. Mycobacterial granulomas from HIV (-) patients showed marginally lower numbers of HLA Class I cells when compared to those from HIV (+) patients. The percentage of FOXP3 positive cells differed significantly between mycobacterial granulomas from HIV (-) and HIV (+) patients. This study highlights the complex interplay between different cell types and cytokines secreted into the microenvironment that ultimately results in containment of the organism or disease progression. Tuberculosis mono-infection causes variation in the expression of cell markers such as FOXP3 with accompanying noteworthy changes in cytokine production in areas of granuloma formation. The alterations noted in TB and HIV co-infection are even greater and point toward evolution of micro-organism synergism with host demise.

MMed, Haematology, Faculty of Health Sciences, University of the Witwatersrand / Background: Mycobacterium tuberculosis (MTB) infection remains a serious public health challenge in sub-Saharan Africa. Rapid and early diagnosis is critical in the successful control of this eminently treatable infection. This study compared the diagnostic usefulness of culture, bone marrow trephine biopsy granulomata, bone marrow trephine biopsy Ziehl-Neelsen (ZN) stain and bone marrow mycobacterial polymerase chain reaction (PCR) in establishing the diagnosis of mycobacterial infection in HIV infected patients. Materials and methods: The trephine biopsies of HIV positive patients done for the investigation of suspected tuberculosis were reviewed for granulomata and stained with ZN stain. The corresponding bone marrow aspirates were subjected to DNA real-time PCR analyses using LightCyler TB Kit® (Roche Diagnostic). Culture results were used as diagnostic gold standard. Results: Of the 60 patients studied, 24 were culture negative. Of the 34 culture positive, 62% were Mycobacterium tuberculosis and 38% were Mycobacterium avium intracellulare. Using the culture method as a gold standard, the sensitivities and specificities were 97% and 23% for bone marrow trephine biopsy granulomata, 65% and 58% for bone marrow trephine biopsy ZN staining and 50% and 73% for bone marrow aspirate PCR analysis respectively. Ninety-seven percent of all trephine biopsies with positive ZN stain had granulomata. Conclusion: The presence of granulomata in bone marrow trephine biopsies of HIV infected patients appear to have a high diagnostic yield whilst mycobacterial PCR has the lowest yield but highest specificity. These results should be confirmed in a prospective case controlled study because the sample size in this study was small, and the study was a retrospective one.

No description available.

A research report Submitted to the faculty of Health Sciences, University of Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Masters of Science in Medicine (Pharmaceutical Affairs). / Pathogenic mycobacterial infection was in recent decades a health concern so well controlled that eradication seemed imminent. However, it is once again reaching epidemic proportions following the increasing prevalence of AIDS. One important means of curbing this resurgence, is a robust method that has the capability of identifying to a species level speciating disease causing mycobacteria in a matter of days. Several new methodologies are now available that enable dramatic reductions in turn-around times. In this study High Performance Liquid Chromatography was investigated to determine how this system compared with the current mycobacterial system of methodologies adopted in South African laboratories. Four species of pathogenic mycobacteria, with a high prevalence in South Africa, were tested in a sample size of 80. Samples were subjected to HPLC, Gene Probes and Biochemical testing. HPLC was the most capable of identifying the mycobacteria to species level displaying a sensitivity to the organisms of 96.25 %. Gene probes and Biochemical testing had sensitivity values of 82.5 % and 80 % respectively. HPLC was also more cost efficient and displayed a wider range of identification. It is therefore suggested that HPLC replace Gene probes and Biochemical testing for purposes of MOTT identification in the comprehensive mycobacterial identification system. The result is a time saving of at least 3 weeks and a cost reduction of approximately 30 %. The large initial capital investment required for the implementation of the HPLC system is justified by the long term cost saving as well as the additional utility derived from early identification. As a consequence treatment is not empiric but rather tailored to the organism infecting the patient, hence preventing multiple drug resistance developing and ultimately saving a life through rational drug use. / WHSLYP2016

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 113-128).

Thematic Paper (M.C.T.M. (Clinical Tropical Medicine))--Mahidol University, 2007. / LICL has E-Thesis 0024 ; please contact computer services. LIRV has E-Thesis 0024 ; please contact circulation services.

Orientador: Antonio Condino Neto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T09:19:02Z (GMT). No. of bitstreams: 1 Prando_CarolinaCardosodeMello_D.pdf: 7021288 bytes, checksum: e93bf24a96be00102eb5a19f5d7c8880 (MD5) Previous issue date: 2008 / Resumo: O sistema NADPH oxidase fagocítico humano possui um papel importante na defesa contra microorganismos intracelulares, incluindo micobactérias. Mutações nas subunidades deste sistema resultam na Doença Granulomatosa Crônica (DGC). O gene CYBB, localizado no cromossomo X, codifica a subunidade gp91phox, e mutações neste gene são responsáveis por cerca de 60% dos casos de DGC. Cerca de 40 anos depois da identificação de DGC, foi identificado o primeiro dos 13 defeitos genéticos associados à Susceptibilidade Mendeliana à Micobacteriose, participantes do eixo IL12/23-IFN-?. Baseado no fato de que ambas as doenças predispõem a infecções por micobactérias e que o IFN-? is é um importante ativador do gene CYBB os autores se propuseram a estudar as características clinicas de pacientes latino-americanos com DGC e o sistema NADPH oxidase e expressão do gene CYBB em pacientes com defeitos no eixo IL-12/23-IFN-?. Em relação às características clínicas: história familiar de infecções graves e/ou de repetição, bem como reação adversa à vacina BCG, linfadenopatia, abscessos de pele e profundos estavam associados à DGC, em comparação com não-DGC avaliados pelo laboratório. Defeitos nos receptores IFNGR1 e IFNGR2 e cadeia B1 do receptor de IL-12 podem apresentar expressão do gene CYBB e atividade do sistema NADPH oxidas e diminuída ou abolida, chegando a níveis comparáveis a um paciente com DGC. O IFN- ? e seus receptores são essenciais para o desenvolvimento e ativação do sistema NADPH oxidase, e pacientes com comprometimento da função deste sistema devem também ser avaliados para defeitos do eixo IL12/23-IFN-? afetando secundariamente o sistema NADPH oxidase. / Abstract: The NADPH phagocytic oxidase system plays a crucial role in host defense against intracellular microorganisms, including mycobacteria. Mutations affecting subunits of this system result in Chronic Granulomatous Disease (CGD). The CYEE gene, located in the X chromosome, encodes gp91 phox, and mutations on this gene account for more than 60% of CGD cases. Almost 40 years after, the first of 13 different genetic disorders associated with Mendelian Susceptibility to Mycobacterial Diseases (MSMD), was identified. The genes responsible for MSMD are part of the IL12/23-IFN-? axis. Based on the fact that both the diseases predispose to mycobacterial infections and that IFN-? is an important activator of CYBB gene, the authors aimed to describe clinical aspects of Latin American CGD patients and investigate ifthe NADPH oxidase system function and gp91phox expression would be affected in patients with defects in the IL-12/23-IFN-? axis. Regarding clinical features familial history of recurrent and sever infections, as well as adverse reactions to BCG vaccine, lymphadenopathy, skin and profound abscess were associated to DGC when compared to clinical features of non-CGD evaluated in the laboratory. Defects of IFNGR1 and IFNGR2 and IL12RB1 may present diminish.ed or abolished gene expression of CYBB and activity of NADPH oxidase system ate levels of a CGD patient. Based on that, we can conclude that IFN- ? and its receptor are essential for development and activation 9f NADPH oxidase system. In addition, patients who present an impaired superoxide release and/or failure on expressing gp91phox should also be evaluated for IL12/23-IFN-? axis affecting secondarily the NADPH oxidase system. / Doutorado / Doutor em Farmacologia

INTRODUÇÃO: A hanseníase é uma doença infectocontagiosa granulomatosa crônica, causada por Mycobacterium leprae e representa ainda um problema de saúde pública no Brasil. Em média 30-40% dos doentes tem risco de desenvolver reações hansênicas, que são considerados estados de exacerbação da resposta imunológica do hospedeiro a antígenos M. leprae. Está bem descrita a importância de células T reguladoras (Tregs) em infecções bacterianas crônicas. Estudos anteriores do nosso grupo demonstraram aumento da proporção de Tregs in situ e em sangue periférico de pacientes multibacilares (mas não em paucibacilares), assim como diminuição da proporção de Tregs na reação tipo 2 (porém não na reação tipo 1), sugerindo a participação deste subtipo celular na imunopatogênese da hanseníase. Entretanto, a baixa frequência de Tregs tem dificultado o estudo da sua capacidade funcional. OBJETIVO: Estabelecer protocolo de expansão de Tregs funcionais em pacientes com hanseníase e, posteriormente, avaliar a capacidade funcional das Tregs expandidas. METODOLOGIA: Células mononucleares do sangue periférico (PBMCs) foram separadas e cultivadas, com anti-CD3 e anti-CD28, acrescidos de IL-2 e rapamicina, por 21 dias. Para avaliar a capacidade funcional das Tregs expandidas, realizou-se um ensaio de co-cultivo das Tregs expandidas e PBMCs autólogas (marcadas com CFSE). Utilizou-se diferentes proporções de Tregs:PBMCs (1:1, 1:2 e 1:5). A casuística foi composta por 29 indivíduos diagnosticados com hanseníase, divididos em quatro grupos, de acordo com a classificação de Ridley e Jopling. Além disso, coletou-se material de 6 indivíduos saudáveis, com a finalidade de caracterizar o pool celular do protocolo de expansão. Para isso, culturas de expansão de indivíduos saudáveis foram submetidas a dois protocolos distintos de purificação celular (beads magnéticas e cell sorting), além de imunofenotipagem das moléculas CD39, PD-1 e LAG3. RESULTADOS: Tregs dos pacientes multibacilar sem reação, nas proporções 1:2 e 1:5, e no grupo reacional tipo 2, na proporção 1:5, mostraram maior capacidade supressora, quando comparados aos grupos pacubacilar sem reação e reacional tipo 2, respectivamente. No grupo de indivíduos saudáveis não houve diferenças na capacidade supressora das Tregs obtidas por separação por beads magnéticas e Tregs obtidas por cell sorting, e não houve expressão significativa de células PD-1+ e LAG-3+ nas culturas de expansão. CONCLUSÃO: Em todos os indivíduos testados, independente da classificação clínica da hanseníase, o protocolo proposto resultou em uma expansão exponencial de uma linhagem de Tregs com atividade supressora, porém com sugestão preliminar de maior eficiência supressora das Tregs na forma MB (com ou sem reação), corroborando a correlação entre atividade supressora de Tregs e maior carga bacilar. Além disso, o protocolo com beads magnéticas se mostrou tão eficaz quanto o protocolo de purificação por cell sorting. A imunofenotipagem das Tregs expandidas de indivíduos saudáveis indicou que não havia número significativo de células exaustas ao fim de nosso protocolo, assim como não houve crescimento concomitante de células T reguladoras do tipo 1 na cultura de expansão / INTRODUCTION: Leprosy is a chronic granulomatous infectious disease caused by Mycobacterium leprae and a public health problem in Brazil. On average, 30-40% of patients are at risk of developing leprosy reactions, which are considered as states of exacerbation of the host\'s immune response to M. leprae antigens. The importance of regulatory T cells (Tregs) in chronic bacterial infections has been well described, but their role in leprosy remains unclear. Previous studies from our group have shown increased proportions of Tregs in situ and in peripheral blood of multibacillary patients (but not in paucibacillary patients), and decrease in the proportion of Tregs in type 2 reaction (but not type 1 reaction), reinforcing the role played by these cells in the immunopathogenesis of leprosy. However, the low numbers of Tregs preclude further studies on their functional capabilities. OBJECTIVE: To establish a protocol for expansion of functional Tregs in patients with leprosy and evaluate the functional capacity of the expanded Tregs. METHOD: Peripheral blood mononuclear cells (PBMCs) were separated and cultured under anti-CD3 and anti-CD28 stimulation, plus IL-2 and rapamycin for 21 days. To evaluate the functional capacity of the expanded Tregs, a co-culture assay of the expanded Tregs and autologous PBMCs (labeled with CFSE) was performed. Different ratios of Tregs: PBMCs (1: 1, 1: 2 and 1: 5) were used. The sample consisted of 29 individuals diagnosed with leprosy, divided into four groups, according to the Ridley and Jopling classification. In addition, samples from 6 healthy individuals were collected to characterize better the cellular characteristics of the expansion protocol. For this, cultures of healthy individuals PBMC were submitted to two distinct cell purification protocols (magnetic beads and cell sorting), as well as immunophenotyping of the molecules CD39, PD-1 and LAG3. RESULTS: Expanded Tregs from the multibacillary patients without reaction, at 1:2 and 1:5 ratios, and with type 2 reaction, at 1: 5 ratio, showed greater suppressive capacity that the respective paucibacillary groups. Tregs from healthy individuals obtained through magnetic beads separation and through cell sorting did not show differences in suppressor capacity, and there was no significant expression of PD-1 + and LAG-3 + on cells of the expansion cultures. CONCLUSION: In all individuals tested, irrespective of the leprosy classification, the proposed protocol resulted in an exponential expansion of Tregs with suppressive capacity; however, preliminary evidence indicated stronger suppressor activity of the Tregs from multibacillary patients (with or without reaction), suggesting a direct correlation between Tregs activity and bacillary load in leprosy. In addition, our protocol with magnetic beads proved to be as effective as the cell sorting protocol. Immunophenotyping of expanded Tregs from healthy subjects revealed that there was no significant number of exhausted cells at the end of our protocol, nor there was the concomitant outgrowth of type 1 regulatory T cells in the expansion protocol

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-03T15:30:41Z No. of bitstreams: 2 Dissertação - Jessyca Rodrigues Braga - 2017.pdf: 4744453 bytes, checksum: 900a3d7f082db5d208a086c58466aa61 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-03T15:31:02Z (GMT) No. of bitstreams: 2 Dissertação - Jessyca Rodrigues Braga - 2017.pdf: 4744453 bytes, checksum: 900a3d7f082db5d208a086c58466aa61 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-03T15:31:02Z (GMT). No. of bitstreams: 2 Dissertação - Jessyca Rodrigues Braga - 2017.pdf: 4744453 bytes, checksum: 900a3d7f082db5d208a086c58466aa61 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-04-04 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Surgical Site Infection (SSI), besides being an indicator of the quality of care, is one of the main healthcare-associated infection (HAI). SSI caused by non-tuberculous mycobacteria (NTM) has become increasingly frequent, increasing the prevalence of SSI in clinical practice. OBJECTIVE: To analyze the epidemiological and clinical aspects of SSI by MNT in the city of Goiânia-Goiás. METHODS: Retrospective cross-sectional study, carried out from the Municipal Coordination of Infection Control and Patient Safety in Health Services (Comciss), of the Municipality of Goiânia - Goiás (Brazil). The population consisted of clinical cases of SSI by probable MNT notified to Comciss, by public and private health institutions of the city of Goiânia-Goiás, in the period from 2006 to 2015. Data were collected during the months of October to December 2015 and March 2016. The source of the data consisted of the notification/investigation forms of SSI cases by MNT stored in Comciss. The data were organized, processed and analyzed in SPSS software version 20.0 and estimated by means of simple frequency and measures of central tendency (average, fashion and median). Chi-square test or Fisher's exact test were used to assess the differences between proportions, and Odds Ratio to estimate the chance of positivity for each outcome. The level of significance was considered p <0.05. RESULTS: One hundred twenty two specimens were positive for MNT and 9 were negative. The average age of the patients was 38.15 years (SD: 13.8, min: 19 and max: 86 years). The female sex predominated in 77.1% of the cases. Comciss was the main responsible (67.1%) for the notifications. The private healthcare institutions accounted for 92.3% of the surgeries performed. Aesthetic surgeries were the most prevalent (51.9%), with mammoplasty being the most frequent. The conventional route stood out with 44.2%, compared to those made by video. The species M. abscessus and M. fortuitum prevailed. Orthopedic surgery (p = 0.002) and general surgery (p <0.001) presented a statistical association for M. abscessus infection. The use of prosthesis (p <0.001, OR 95%: 6.12) and plastic surgery (p <0.001; OR 95%: 5.90) showed statistical difference for M. fortuitum infection, but were not associated in regression. Signs / symptoms were present in 119 patients, including fever, secretion, abscess, nodules, hyperemia, among others. Clarithromycin was the most commonly used antimicrobial for the treatment of SSI by M. abscessus, and amikacin-associated clarithromycin was the most commonly used antimicrobial therapy for M. fortuitum. Resistance to 12 (80%) different antimicrobials was found for M. abscessus, and 15 (88.2%) for M. fortuitum. CONCLUSIONS: Females predominated, with mammoplasty being the main surgery. M. abscessus and M. fortuitum were the most prevalent species, being resistant to up to 15 (88,2%) antimicrobials. Being submitted to orthopedic and general surgery was a factor associated with ISC by M. abscessus. / A Infecção de Sítio Cirúrgico (ISC), além de ser um indicador da qualidade da assistência, é uma das principais infecções relacionadas à assistência à saúde (IRAS). As ISC causadas pelas Micobactérias Não Tuberculosas (MNT) têm se tornado cada vez mais frequentes, aumentando a prevalência de ISC na prática clínica. OBJETIVO: Analisar os aspectos epidemiológicos e clínicos das ISC por MNT no município de Goiânia-Goiás. MÉTODO: Estudo retrospectivo do tipo transversal, realizado a partir do banco de dados da Coordenação Municipal de Controle de Infecção e Segurança do Paciente em Serviços de Saúde (Comciss) do Município de Goiânia – Goiás (Brasil). A população constituiu-se de casos clínicos de ISC por provável MNT notificados à Comciss por instituições de saúde pública e privadas do município de Goiânia-Goiás, no período de 2006 à 2015. Os dados foram coletados durante os meses de outubro a dezembro de 2015 e março de 2016. A fonte dos dados constituiu-se das fichas de notificação/investigação dos casos de ISC por MNT armazenadas na Comciss. Os dados foram organizados, processados e analisados no software SPSS versão 20.0 e estimados por meio de frequência simples e medidas de tendência central (média, moda e mediana). Teste de Qui- quadrado ou exato de Fischer foram utilizados para avaliar as diferenças entre proporções, e Odds Ratio para estimar a chance de positividade para cada desfecho. O nível de significância foi considerado p<0,05. RESULTADOS: Foram positivas 122 espécimes para MNT e 9 negativos. A média de idade dos pacientes foi 38,15 anos (DP: 13,8; min:19 e máx: 86 anos). Predominou o sexo feminino em 77,1% dos casos. A Comciss foi a principal responsável (67,1%) pelas notificações. As instituições de saúde da rede privada foram responsáveis por 92,3% das cirurgias realizadas. As cirurgias estéticas foram as mais prevalentes (51,9%), sendo a mamoplastia a mais frequente. A via convencional se sobressaiu, com 44,2% em relação às realizadas por vídeo. Predominaram as espécies M. abscessus e M. fortuitum. Cirurgia ortopédica (p=0,002) e cirurgia geral (p<0,001) apresentaram associação estatística para infecção pelo M. abscessus. O uso de prótese (p<0,001; OR 95%: 6,12) e cirurgia plástica (p<0,001; OR 95%: 5,90) mostraram diferença estatística para infecção por M. fortuitum, porém não apresentaram associação nos modelos de regressão. Cento de dezenove pacientes apresentaram sinais/sintomas, verificou-se febre, secreção, abscesso, nódulos, hiperemia, dentre outros. Claritromicina foi o antimicrobiano mais utilizado para tratamento de ISC por M. abscessus, e claritromicina associada à amicacina foi terapêutica antimicrobiana mais utilizada para M. fortuitum. Encontrou-se resistência a 12 (70,0%) diferentes antimicrobianos para M. abscessus, e a 15 (88,2%) para M. fortuitum. CONCLUSÕES: Predominou o sexo feminino, sendo a mamoplastia a principal cirurgia envolvida. M. abscessus e M. fortuitum foram as espécies mais prevalentes, sendo as mesmas resistentes à até 15 (88,2%) antimicrobianos. Ser submetido à cirurgia ortopédica e geral foi fator associado à ISC por M. abscessus.

Over the past several decades, the rate of pulmonary nontuberculous my- cobacterial (PNTM) disease has been increasing. PNTM patients gener- ally consist of lean and tall women presenting with symptoms in the sixth decade of life. They have a de nitive morphophenotype, but no consistent immunological abnormalities despite extensive investigation. I hypothesized that respiratory epithelial dysfunction might play a critical role in PNTM disease predisposition because diseases with defects of mucociliary transport have high rates of PNTM disease that increase with age, suggesting a direct connection between airway epithelial function and PNTM disease. I found that PNTM patients have a distinct respiratory epithelial phenotype ex vivo and decreased nasal nitric oxide levels in vivo. The PNTM ex vivo phenotype consists of an abnormally low resting ciliary beat frequency (CBF) and abnormal CBF response to toll-like receptor (TLR) agonists. The depressed baseline CBF response in PNTM patient cells can be normalized ex vivo by augmenting the nitric oxide-cyclic guanosine monophosphate pathway without appreciable e ect on CBF in healthy controls. In healthy controls, bacterial TLR agonists increase CBF and viral TLR agonists decrease CBF. In PNTM patients these responses are impaired and are not normalized with the normalization of the resting CBF rate. Inhibitor-induced disruption of signalling pathways associated with CBF regulation demonstrated that the majority of the CBF response to TLR agonists involves the PI-3K pathway and PKC. Inhibition of the PI-3K pathway (PI-3K , Akt1, and PDK1) closely mimicked the ex vivo phenotype seen in PNTM patient respiratory epithelia. These data identify a novel aspect of PNTM disease with in vivo and ex vivo correlates that suggest that PNTM infection is associated with abnormal function at both the CBF and TLR response levels. This phenotype is novel, reproducible, and provide a foundation with which to determine the genetic basis of PNTM infection.